Polypeptide and peptide therapeutics are widely used in medical practice. Their ease of production, either by recombinant DNA technology or peptide synthesizers, ensures their continued use in a variety of circumstances in the years to come. Accordingly, polypeptide therapeutics, such as hormones, cytokines and growth factors, represent an important class of therapeutic agents. Certain native polypeptides, however, can be inactivated rapidly in vivo via proteolysis or isomerization. Such inactivation can be inconvenient in cases where it is desired to maintain a consistent or sustained blood level of the therapeutic over a period of time, as repeated administrations are then necessary. In certain instances, one or more of the proteolytic products of the polypeptide can be antagonistic to the activity of the intact polypeptide. In these cases, administration of additional therapeutic alone may be insufficient to overcome the antagonist effect of the proteolytic products.
To further illustrate, one class of peptide hormones whose prolonged presence in the blood may be beneficial include glucagon-like peptides 1 and 2 (GLP-1 and GLP-2 respectively), glucose-dependent insulinotropic peptide (GIP), neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY). GLP-1 is an important polypeptide hormone with regulatory function in glucose metabolism and gastrointestinal secretion and metabolism. Current efforts show that GLP-1 is a growth factor for beta cells in the pancreas and perhaps is involved in cell differentiation in other organs as well. GLP-2 is a 33-amino acid peptide having therapeutic application in the treatment of diseases of the gastrointestinal tract. In particular, it has been determined that GLP-2 acts as a trophic agent to enhance and maintain proper gastrointestinal function, as well as to promote growth of intestinal tissues (See, e.g., U.S. Pat. Nos. 5,834,428; 5,789,379; and 5,990,077; and International Publication No. WO 98/52600). GIP is a 42-amino acid peptide synthesized and secreted from endocrine cells in the small intestine (See R. A. Pederson, et al., Endocrinology 99, 780-785 (1976) and T. B. Usdin, et al., Endocrinology 133, 2861-2870 (1993)). GIP infusions have been shown to inhibit the effects of glucagon on the liver while enhancing those of insulin. Additionally, GIP has dual effects on hepatic blood flow, increasing flow through the portal vein and inhibiting flow through the hepatic artery. Neuropeptide Y is a 36-amino acid member of the pancreatic polypeptide family. It is highly concentrated in both the central and peripheral mammalian nervous system, is the most potent substance known to cause an increase in feeding, and may play a role in the genetic basis of Type II Diabetes Mellitus (See U.S. Pat. Nos. 6,410,701, 6,075,009, 5,026,685, 5,328,899, and K. Tatemoto, Proc. Natl. Acad. Sci. USA 79, 5485-5489 (1982)). Peptide YY (PYY) and pancreatic polypeptide (PP) are structurally related peptide hormones involved in memory loss, depression, anxiety, epilepsy, pain, hypertension, and sleep and eating disorders.
These polypeptide hormones, and other polypeptide factors, are believed to be degraded by members of the post-proline cleaving class of serine proteinase enzymes, such as dipeptidyl peptidase IV (DPP IV). DPP IV is a membrane associated serine peptidase which cleaves N-terminal dipeptides from a peptide chain containing in the penultimate (P1) position, preferably, a proline residue, or an alanine residue if the N-terminal residue (P2) is histidine or a large aromatic such as tyrosine, tryptophan or phenylalanine. The amino terminus sequences of GLP-1, GIP, and GLP-2 are His-Ala-Glu, Tyr-Ala-Glu, and His-Ala-Asp respectively. The amino terminal sequences of NPY, PP, and PYY are Tyr-Pro-Ser, Ala-Pro-Leu and Tyr-Pro-Ile respectively. Hence, DPP IV has been implicated in the regulation of the activity of each of these polypeptide hormones, as well as other polypeptides, in vivo.
DPP IV-mediated removal of Xaa-Ala or Xaa-Pro dipeptides, wherein Xaa is an amino acid residue, from the N-terminus of the bioactive peptide hormones mentioned above renders them inactive, or even antagonistic. Accordingly, cleavage and inactivation of peptide hormones by serine proteinases such as DPP IV is just one example that illustrates the significant limitation imposed by proteolysis for the use of therapeutic polypeptides. The discovery of analogs that exhibit stability towards proteolysis, such as DPP IV-mediated inactivation, is therefore of substantial interest. Accordingly, there is a need in the art for proteolysis-resistant peptide hormones.